Compositions of treating hepatitis virus infections with N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds in combination therapy

ABSTRACT

Provided are methods and compositions for treating hepatitis virus infections in mammals, especially humans. The methods comprise (1) administering N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds in combination with nucleoside antiviral agents, nucleotide antiviral agents, mixtures thereof, or immunomodulating/immunostimulating agents, or (2) administering N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds in combination with nucleoside antiviral agents, nucleotide antiviral agents, or mixtures thereof, and immunomodulating/immunostimulating agents.

RELATED APPLICATION DATA

This application is a continuation of the application Ser. No.09/023,401 filed Feb. 12, 1998, which claims priority to US provisionalapplication No. 60/041,221 filed Feb. 14, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and compositions for treatinghepatitis virus infections, especially hepatitis B virus infections, inmammals, especially humans. The methods comprise (1) administeringN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds in combinationwith nucleoside antiviral-agents, nucleotide antiviral agents, mixturesthereof, or immunomodulating/-immunostimulating agents, or (2)administering N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compoundsin combination with nucleoside antiviral agents, nucleotide antiviralagents, or mixtures thereof, and immunomodulating/immunostimulatingagents. Such combinations of anti-hepatitis viral agents show unexpectedefficacy in inhibiting replication and secretion of hepatitis viruses incells of mammals infected with these viruses.

2. Description of Related Art

Hepatitis Viruses

Hepatitis B Virus (HBV, HepB) is a causative agent of acute and chronicliver disease including liver fibrosis, cirrhosis, inflammatory liverdisease, and hepatic cancer that can lead to death in some patients(Joklik, Wolfgang K., Virology, Third Edition, Appleton & Lange,Norwalk, Conn., 1988 (ISBN 0-8385-9462-X)). Although effective vaccinesare available, there are still more than 300 million people worldwide,i.e., 5% of the world's population, chronically infected with the virus(Locarnini, S. A., et. al., Antiviral Chemistry & Chemotherapy (1996)7(2):53-64). Such vaccines have no therapeutic value for those alreadyinfected with the virus. In Europe and North America, between 0.1% to 1%of the population is infected. Estimates are that 15% to 2.0% ofindividuals who acquire the infection develop cirrhosis or anotherchronic disability from HBV infection. Once liver cirrhosis isestablished, morbidity and mortality are substantial, with about a5-year patient survival period (Blume, H., E., et. al., Advanced DrugDelivery Reviews (1995) 17:321-331). It is therefore necessary and ofhigh priority to find improved and effective anti-HBV anti-hepatitistherapies (Locarnini, S. A., et. al., Antiviral Chemistry & Chemotherapy(1996) 7(2): 53-64).

Other hepatitis viruses significant as agents of human disease includeHepatitis A, Hepatitis B, Hepatitis C, Hepatitis Delta, Hepatitis E,Hepatitis F, and Hepatitis G (Coates, J. A. V., et. al., Exp. Opin.Ther. Patents (1995) 5(8):747-756). In addition, there are animalhepatitis viruses that are species-specific. These include, for example,those infecting ducks, woodchucks, and mice.

1,5-dideoxy-1,5-imino-D-glucitol Compounds

1,5-dideoxy-1,5-imino-D-glucitol (also known as 1-deoxynojirimycin, DNJ)and its N-alkyl derivatives are known inhibitors of the N-linkedoligosaccharide processing enzymes alpha glucosidase I and II (Saunieret al., J. Biol. Chem. (1982) 257:14155-14161 (1982); Elbein, Ann. Rev.Biochem. (1987) 56:497-534). As glucose analogs, they also havepotential to inhibit glucose transport, glucosyl-transferases, and/orglycolipid synthesis (Newbrun et al., Arch. Oral Biol. (1983) 28:516-536; Wang et al., Tetrahedron Lett. (1993) 34:403-406). Theirinhibitory activity against glucosidases has led to the development ofthese compounds as anti-hyperglycemic agents and antiviral agents. See,for example, PCT International Publication WO 87/03903 and U.S. Pat.Nos. 4,065,562; 4,182,767; 4,533,668; 4,639,436; 4,849,430; 4,957,926;5,011,829; and 5,030,638.

Glucosidase inhibitors such as N-alkyl-1,5-dideoxy-1,5-imino-D-glucitolcompounds wherein the alkyl group contains between three and six carbonatoms have been shown to be effective in the treatment of Hepatitis Binfection (PCT International Publication WO 95/19172). For example,n-butyl-deoxynojirimycin (n-butyl-DNJ;N-butyl-1-5-dideoxy-1,5-imino-D-glucitol) is effective for this purpose(Block, T. M., Proc. Natl. Acad. Sci. USA (1994) 91:2235-2239; Ganem, B.Chemtracts: Organic Chemistry (1994) 7(2), 106-107). N-butyl-DNJ hasalso been tested as an anti-HIV-1 agent in HIV infected patients, and isknown to be well tolerated. Another alpha glucosidase inhibitor,deoxynojirimycin (DNJ), has been suggested as an antiviral agent for usein combination with N-(phosphonoacetyl)-L-aspartic acid (PALA) (WO93/18763). However, combinations of N-substituted-imino-D-glucitolderivatives and other antiviral agents for the treatment of hepatitisvirus infections have not been previously disclosed or suggested.

Nucleoside and Nucleotide Antiviral Agents

Reverse transcriptase inhibitors, including the class of nucleoside andnucleotide analogs, were first developed as drugs for the treatment ofretroviruses such as human immunodeficiency virus (HIV), the causativeagent of AIDS. Increasingly, these compounds have found use againstother viruses, including both RNA and DNA viruses, via viral screeningand chemical modification strategies. Nucleoside and nucleotide analogsexert their antiviral activities by inhibiting the corresponding DNA andRNA polymerases responsible for synthesis of viral DNA and RNA,respectively. Because viruses contain different forms of polymerases,the same nucleoside/nucleotide compound can have a dramaticallydifferent effect against different viruses. For example, lamivudine(3TC™) appears to be useful against HBV infection, whereas zidovudine(AZT™) appears to have little use against the same virus (Gish, R. G.,et al., Exp. Opin. Invest. Drugs (1995) 4(2):95-115)

Toxicity has been significant with some nucleoside analog antivirals.For example, clinical tests on the use of the nucleoside analogfialuridine (FIAU) for treatment of chronic hepatitis B were suspendedrecently due to drug-related liver failure leading to death in somepatients. Consequently, there is still a need for safer drug regimensfor the treatment of hepatitis B infections and hepatitis (Mutchnick, M.G., et. al., Antiviral Research (1994) 24:245-257).

Immunomodulators and Immunostimulants

Immunomodulators/immunostimulators such as interferon alfa and othercytokines have been-used for the treatment of HBV infection withpromising results. Unfortunately, the response rates are lower thandesired. Interferon treatment is currently approved by the FDA for thetreatment of Hepatitis B. Other immune system-affecting drug candidatesare presently being investigated. These include thymic pepides for usein the treatment of chronic hepatitis B (CHB), isoprinosine, steroids,Shiff base-forming salicylaldehyde derivatives such as Tucaresol,levamisol, and the like (Gish, R. G., et. al., Exp. Opin. Invest. Drugs(1995) 4(2):95-115; Coates, J. A. V., et. al., Exp. Opin. Ther. Patents(1995) 5(8):747-765).

The use of N-substituted-imino-D-glucitol compounds in combination withimmunomodulating/immunostimulating agents is novel.

SUMMARY OF THE INVENTION

As noted above, the combination of N-substituted-imino-D-glucitolcompounds and derivatives thereof with other anti-hepatitis viruscompounds has, to the present inventor's knowledge, neither beensuggested nor disclosed. The use of two or more anti-viral agents toprovide improved therapy for the treatment of hepatitis B virusinfections is desirable due to the morbidity and mortality of thedisease. Combination therapy is also desirable since it should reducetoxicity in patients as it enables the physician to administer lowerdoses of one or more of the drugs being given to a patient. Combinationtherapy can also help to prevent the development of drug resistance inpatients (Wiltink, E. H. H., Pharmaceutish Weekblads Scientific Edition(1992) 14(4A):268-274). The result of an improved efficacy configurationcombined with a relative lack of toxicity and development of resistancewould provide a much improved drug treatment profile.

The present inventor has surprisingly discovered that the combined useof N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds andnucleoside or nucleotide antiviral compounds, or combinations thereof,and/or immunomodulators/immunostimulants, results in unexpectedlygreater anti-hepatitis virus effectiveness of the compounds compared tothe combined antiviral activities expected of the individual compoundsalone. Whether this is due to different mechanisms of action of thedifferent classes of drugs employed or some other biological phenomenonis presently unclear.

Accordingly, in a first aspect, the present invention provides a methodof treating a hepatitis virus infection in a mammal, comprisingadministering to said mammal a first amount of anN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula I:

wherein:

R is selected from the group consisting of arylalkyl, cycloalkylalkyl,and branched or straight chain alkyl having a chain length of C₁ to C₂₀,and

W, X, Y, and Z are each independently selected from the group consistingof hydrogen, alkanoyl, aroyl, and trifluoroalkanoyl; and

a second amount of an antiviral compound selected from the groupconsisting of a nucleoside antiviral compound, a nucleotide antiviralcompound, and mixtures thereof,

wherein said first and second amounts of said compounds togethercomprise an anti-hepatitis virus effective amount of said compounds.

In a second aspect, the present invention provides a method of treatinga hepatitis B virus infection in a mammal, comprising administering tosaid mammal from about 0.1 mg/kg/day to about 100 mg/kg/day of anN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula I, asabove, and from about 0.1 mg/person/day to about 500 mg/person/day of acompound selected from the group consisting of a nucleoside antiviralcompound, a nucleotide antiviral, and mixtures thereof.

The present invention also provides a method of treating a hepatitis Bvirus infection in a human patient, comprising administering to saidhuman patient from about 0.1 mg/kg/day to about 100 mg/kg/day ofN-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol and from about 0.1mg/person/day to about 500 mg/person/day of(−)-2′-deoxy-3′-thiocytidine-5′-triphosphate.

Also provided is a composition, comprising anN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula I, asabove, and an antiviral compound selected from the group consisting of anucleoside antiviral compound, a nucleotide antiviral compound, andmixtures thereof.

The invention also provides a pharmaceutical composition, comprising afirst amount of an N-substituted-1,5-dideoxy-1,5-imino-D-glucitolcompound of Formula I, as above, and a second amount of an antiviralcompound selected from the group consisting of a nucleoside antiviralcompound, a nucleotide antiviral compound, and mixtures thereof, as wellas a pharmaceutically acceptable carrier, diluent, or excipient.

The invention further provides a pharmaceutical composition for treatinga hepatitis B virus infection in a mammal, comprising from about 0.1 mgto about 100 mg of an N-substituted-1,5-dideoxy-1,5-imino-D-glucitolcompound of Formula I, as above, and from about 0.1 mg to about 500 mgof a compound selected from the group consisting of a nucleosideantiviral compound, a nucleotide antiviral, and mixtures thereof.

Also provided is a pharmaceutical composition for treating a hepatitis Bvirus infection in a human patient, comprising from about 0.1 mg toabout 100 mg of N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol, and fromabout 0.1 mg to about 500 mg of(−)-2′-deoxy-3′-thiocytidine-5′-triphosphate.

In addition to the foregoing, the present invention also providesmethods and compositions like those listed above, wherein theN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds of Formula I,nucleoside antiviral compounds, nucleotide antiviral compounds, ormixtures of nucleoside and nucleotide antiviral compounds, are used incombination with immunomodulators, immunostimulators, or mixtures ofimmunomodulators and immunostimulators.

In another aspect, the present invention also provides methods andcompositions like those listed before the paragraph immediately above,wherein the N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds ofFormula I are used in combination with immunomodulators,immunostimulators, or mixtures of immunomodulators andimmunostimulators, but without nucleoside antiviral compounds,nucleotide antiviral compounds, or mixtures of nucleoside and nucleotideantiviral compounds.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below.However, it should be understood that the following detailed descriptionand examples, while indicating preferred embodiments of the invention,are given by way of illustration only since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features, and advantages of the presentinvention will be better understood from the following detaileddescription taken in conjunction with the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention, in which:

FIG. 1 shows the anti-hepatitis B virus activity of(−)-2′-deoxy-3′-thiocytidine-5′-triphosphate (3TC) alone and incombination with N-nonyl-DNJ in vitro.

FIG. 2 shows the plasma concentration of N-nonyl-DNJ versus dose ofN-nonyl-DNJ for each animal in Example 4, from samples taken duringdosing. Animals are indicated by unique letters, and a small amount ofrandom noise has been added to the dose value so that overlapping valuescan be distinguished.

FIG. 3 shows the slope of Log(IPDNA+10) to week versus dose. A distinctletter is used for each animal. The fitted line is from a four parameterlogistic model. The parameters of the fitted curve and their approximatestandard errors are shown on the plot.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is provided to aid those skilled inthe art in practicing the present invention. Even so, this detaileddescription should not be construed to unduly limit the presentinvention as modifications and variations in the embodiments discussedherein can be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

The contents of each of the references cited herein, including thecontents of the references cited within these primary references, areherein incorporated by reference in their-entirety.

The present inventor has discovered that combinations ofN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds withanti-hepatitis virus nucleoside or nucleotide analogs, and/orimmunomodulators/-immunostimulants, are more effective in inhibitinghepatitis virus replication than that which would be expected via thecombined use of the individual compounds.

The present invention thus provides pharmaceutical compositions andmethods of treating hepatitis virus infections, especially hepatitis Bvirus infections, in humans, other mammals, and cells using acombination of an N-substituted-1,5-dideoxy-1,5-imino-D-glucitolcompound with either an antiviral nucleoside, an antiviral nucleotide,mixtures thereof, and/or an immunomodulating or immunostimulating agent.The N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds have basicnitrogen atoms and may be used in the form of a pharmaceuticallyacceptable salt. Nucleosides and nucleotides useful in the presentinvention are substituted purine or pyrmidine heterocycles furthersubstituted with R¹ at the 9 position in the case of purines or with R¹at the 1 position in the case of pyrimidines. The immunomodulating andimmunostimulating agents useful in the present invention include thosethat stimulate immune responses effective in controlling or eliminatingviruses or other infectious agents. Non-limiting examples of suchimmunomodulating and immunostimulating agents include cytokines, peptideagonists, steroids, and classic drugs such as levamisol. The drugcombinations of this invention may be provided to a cell or cells, or toa human or other mammalian patient, either in separate pharmaceuticallyacceptable formulations, formulations containing more than onetherapeutic agent, or by an assortment of single agent and multipleagent formulations. However administered, these drug combinations forman anti-hepatitis virus effective amount of components.

As used herein, the term “anti-hepatitis-virus effective amount” refersto a combined amount of (1) anN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound with either anantiviral nucleoside, an antiviral nucleotide, a mixture of an antiviralnucleoside and an antiviral nucleotide, or animmunomodulating/-immunostimulating agent (or mixtures thereof), or (2)a combined amount of an N-substituted-1,5-dideoxy-1,5-imino-D-glucitolcompound with an antiviral nucleoside, an antiviral nucleotide, or amixture thereof, and an immunomodulating/-immunostimulating agent (ormixtures thereof) effective in treating hepatitis virus infection. Theantiviral effectiveness of the aforementioned combinations may involve avariety of different phenomena associated with viral replication andassembly. These may include, for example, blocking hepatitis viral DNAsynthesis; blocking viral transcription; blocking virion assembly;blocking virion release or secretion from infected-cells; blocking oraltering viral protein function, including the function of viralenvelope protein(s); and/or the production of immature or otherwisenon-functional virions. The overall effect is an inhibition of viralreplication and infection of additional cells, and therefore inhibitionof the progress of infection in the patient.

N-substituted-1,5-dideoxy-1,5-imino-D-glucose Compounds

N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds useful in thepresent invention are represented by structure I below:

wherein R is selected from arylalkyl, cycloalkylalkyl, and branched orstraight chain alkyl having a chain length of C₁ to C₂₀, preferably C₂to C₁₄, more preferably C₆ to C₁₂. R can also be C₁ to C₂₀ alkyl,preferably C₂ to C₁₄, more preferably C₆ to C₁₂, containing 1 to 5, morepreferably 1 to 3, most preferably 1 to 2, oxygen atoms, i.e., oxaderivatives. Preferred R oxa derivatives are 3-oxanonyl, 3-oxadecyl,7-oxanonyl, and 7-oxadecyl. W, X, Y and Z are independently selectedfrom hydrogen, alkanoyl, aroyl, and trifluoroalkanoyl.

Representative N-substituted-imino-D-glucitol compounds useful in thepresent invention include, but are not limited to:

-   N-(n-hexyl-)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(n-heptyl-)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(n-octyl-)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(n-octyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(n-decyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(n-undecyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(n-decyl-)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(n-undecyl-)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(n-dodecyl-)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(2-ethylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(4-ethylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(5-methylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(3-propylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(1-pentylpentylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(1-butylbutyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(7-methyloctyl-)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(8-methylnonyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(9-methyldecyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(10-methylundecyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(6-cyclohexylhexyl-)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(4-cyclohexylbutyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(2-cyclohexylethyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(1-cyclohexylmethyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(1-phenylmethyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(3-phenylpropyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(3-(4-methyl)-phenylpropyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(6-phenylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;-   N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(n-decyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(n-undecyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(n-dodecyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(2-ethylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(4-ethylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(5-methylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(3-propylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(1-pentylpentylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol,    tetrabutyrate;-   N-(1-butylbutyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(7-methyloctyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(8-methylnonyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(9-methyldecyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(10-methylundecyl)-1,5-dideoxy-1,5-imino-D-glucitol,    tetrabutyrate;-   N-(6-cyclohexylhexyl-)-1,5-dideoxy-1,5-imino-D-glucitol,    tetrabutyrate;-   N-(4-cyclohexylbutyl)-1,5-dideoxy-1,5-imino-D-glucitol,    tetrabutyrate;-   N-(2-cyclohexylethyl)-1,5-dideoxy-1,5-imino-D-glucitol,    tetrabutyrate;-   N-(1-cyclohexylmethyl)-1,5-dideoxy-1,5-imino-D-glucitol,    tetrabutyrate;-   N-(1-phenylmethyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(3-phenylpropyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate;-   N-(3-(4-methyl)-phenylpropyl)-1,5-dideoxy-1,5-imino-D-glucitol,    tetrabutyrate; and-   N-(6-phenylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate.

Preferred compounds are N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitoland N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate.

The N-substituted-imino-D-glucitol compounds useful in the presentinvention can be prepared by methods well known in the art as describedin, for example, U.S. Pat. Nos. 4,182,767, 4,639,436, and 5,003,072, aswell as PCT International Publication WO 95/19172 and the referencescited therein. Methods for introducing oxygen into alkyl side chains aredisclosed in Tan et al., (1994) Glycobiology 4(2):141-149. Non-limitingillustrative preparation procedures are presented below in Examples 1and 2.

In treating hepatitis virus infections, one can use the anti-hepatitisvirus combinations or individual compounds of this invention in the formof salts derived from inorganic or organic acids. These salts includebut are not limited to the following: acetate, adipate, alginate,citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,camphorate, camphorsulfonate, digluconate, cyclopentanepropionate,dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, mesylate, andundecanoate.

The basic nitrogen-containing groups can be quaternized with agents suchas lower alkyl halides, such as methyl, ethyl, propyl, and butylchloride, bromides, and iodides; dialkyl sulfates such as dimethyl,diethyl, dibuytl, and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl, and stearyl chlorides, bromides, and iodides; aralkylhalides such as benzyl and phenethyl bromides, and others. Water- oroil-soluble or dispersible products are thereby obtained as desired. Thesalts are formed by combining the basic compounds with the desired acid.

Other compounds of the combinations of this invention that are acids canalso form salts. Examples include salts with alkali metals or alkalineearth metals, such as sodium, potassium, calcium, or magnesium, or withorganic bases or basic quaternary ammonium salts.

Compounds of the combinations of this invention may be acids or bases.As such, they may be used to form salts with one another. For example,the phosphoric acid form of (−)-2′-deoxy-3′-thiocytidine-5′-triphosphatewill form a salt with the base form ofN-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol orN-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate. This typeof salt can then be provided to the patient in a pharmaceuticallyacceptable formulation or as a pure single salt.

In some cases, the salts can also be used as an aid in the isolation,purification, or resolution of the compounds of this invention.

Nucleosides and Nucleotides

Nucleosides and nucleotides useful in the present invention are purine(II) base compounds or pyrimidine (III) base compounds, or analogs suchas compounds IV or V.

Position numbering for purines and pyrmidines is as shown in structuresII and III. R can be selected from hydroxyalkyl, hydroxyalkenyl,thiolalkyl, alkylthioalkyl, alkoxyalkyl, alkoxyalkenyl, heterocycle,heterocycloalkyl, hydroxyalkylalkoxyalkyl, alkoxyalkylalkoxyalkyl, andcycloalkylalkyl. The purine compounds can be further substituted atpositions 1, 2, 3, 6, 7, or 8 of the purine heterocycle, and thepyrimidine compounds can be substituted at positions 2, 3, 4, 5, or 6 ofthe pyrimidine heterocycle. Such substituents can be selected fromhydroxy, alkoxy, halo, thiol, amino, mono-substituted amino,di-substituted amino, and alkyl.

When used in combination with another radical when referring to thepurines and pyrimidines useful in the present invention, the term“alkyl” means a straight or branched chain hydrocarbon radicalcontaining from 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms.When used in combination with another radical, the term “alkenyl” meansa straight or branched chain hydrocarbon radical having 1 or more doublebonds, containing from 2 to 8 carbon atoms, preferably 1 to 4 carbonatoms. When used alone when referring to purines and pyrimidines usefulin the present invention, the term “alkyl” means a straight or branchedchain alkyl radical containing from six to 14 carbon atoms, preferablyseven to 12 carbon atoms, and most preferably eight to 11 carbon atoms.The term “aryl” alone or in combination with another radical means aphenyl, naphthyl, or indenyl ring, optionally substituted with one ormore substituents selected from alkyl, alkoxy, halogen, hydroxy, ornitro. “Alkanoyl” means branched or straight chain alkanecarbonyl havinga chain length of C₁ to C₂₀, preferably C₂ to C₁₄, more preferably C₄ toC₁₀; “aroyl” means arylcarbonyl; and “trifluoroalkanoyl” means alkylcontaining three fluoro substituents. “Halogen” means fluorine,chlorine, bromine, or iodine. “Thiol” means sulfur substituted withhydrogen (—SH). “Amino” means nitrogen with two hydrogen atoms;“monosubstituted amino” and “disubstituted amino” mean amino groupsfurther independently substituted with one or more alkyl or arylalkylgroups. “Hydroxyalkyl” means an alkyl group substituted with one or morehydroxyl groups; “hydroxyalkenyl” means an alkenyl group substitutedwith one or more hydroxyl groups; “thioalkyl” means an alkyl substitutedwith one or more thiol (SH) groups; “alkoxyalkyl” means an alkylsubstituted with one or more alkyl ether groups; “alkoxyalkenyl” meansan alkenyl group substituted with one or more alkyl ether groups;“hydroxyalkylalkoxyalkyl”, means an alkoxyalkyl group substituted with ahydroxyalkyl group; “alkoxyalkylalkoxyalkyl” means an alkoxyalkyl groupsubstituted with an alkoxyalkyl group; “cycloalkylalkyl” means an alkylgroup substituted with a cycloalkyl group. The term “heterocycle,” aloneor in combination, means a saturated or partially unsaturated 5 or6-membered ring containing one or more oxygen, nitrogen, and/or sulfurheteroatoms. Said heterocycle can further be substituted with one tofour substituents, which can be independently, hydroxy, hydroxyalkyl,thiol, alkoxy, azido, nitro, a halogen atom, amino, mono-substitutedamino, or disubstituted amino. Heterocycloalkyl means an alkyl groupwherein one or more hydrogen atoms are replaced by a substituted orunsubstituted heterocyclic ring.

Also included are the tautomers of the substituents on the compounds ofthe invention. Non-limiting examples of tautomers are ketone/enoltautomers, imino/amino tautomers, N-substituted imino/N-substitutedamino tautomers, thiol/thiacarbonyl tautomers, and ring-chain tautomerssuch as the five and six membered ring oxygen, nitrogen, sulfur, oroxygen- and sulfur-containing heterocycles also containing substituent-salpha to the heteroatoms. Also specifically included in the presentinvention are enantiomers and diastereomers, as well as racemates andisomeric mixtures of the compounds discussed herein.

Representative nucleoside and nucleotide compounds useful in the presentinvention include, but are not limited to:

-   (+)-cis-5-fluoro-1-[2-(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosine;-   (−)-2′-deoxy-3′-thiocytidine-5′-triphosphate (3TC);-   (−)-cis-5-fluoro-1-[2-(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosine    (FTC);-   (−)-2′,3′, dideoxy-3′-thiacytidine [(−)-SddC];-   1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine    (FIAC);-   1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine    triphosphate (FIACTP);-   1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-methyluarcil    (FMAU);-   1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide;-   2′,3′-dideoxy-3′-fluoro-5-methyl-dexocytidine (FddMeCyt);-   2′,3′-dideoxy-3′-chloro-5-methyl-dexocytidine (ClddMeCyt);-   2′,3′-dideoxy-3′-amino-5-methyl-dexocytidine (AddMeCyt);-   2′,3′-dideoxy-3′-fluoro-5-methyl-cytidine (FddMeCyt);-   2′,3′-dideoxy-3′-chloro-5-methyl-cytidine (ClddMeCyt);-   2′,3′-dideoxy-3′-amino-5-methyl-cytidine (AddMeCyt);-   2′,3′-dideoxy-3′-fluorothymidine (FddThd);-   2′,3′-dideoxy-beta-L-5-fluorocytidine (beta-L-FddC);-   2′,3′-dideoxy-beta-L-5-thiacytidine;-   2′,3′-dideoxy-beta-L-5-cytidine (beta-L-ddC);-   2′-deoxy-3′-thia-5-fluorocytosine;-   3′-amino-5-methyl-dexocytidine (AddMeCyt);-   3′-azido-3′-deoxythymidine (AZT);-   3-chloro-5-methyl-dexocytidine (ClddMeCyt);-   9-(2-phosphonyl-methoxyethyl)-2′,6′-diaminopurine-2′,3′-dideoxyriboside;-   9-(2-phosphonylmethoxyethyl)adenine (PMEA);-   acyclovir triphosphate (ACVTP);-   D-carbocyclic-2′-deoxyguanosine (CdG);-   dideoxy-cytidine;-   dideoxy-cytosine (ddC);-   dideoxy-guanine (ddG);-   dideoxy-inosine (ddI);-   E-5-(2-bromovinyl)-2′-deoxyuridine triphosphate;-   fluoro-arabinofuranosyl-iodouracil;-   stavudine;-   2-deoxy-3′-thia-5-fluorocytidine;-   2′,3′-dideoxy-guanine; and-   2′,3′-dideoxy-guanosine.

A preferred compound is (−)-2′-deoxy-3′-thiocytidine-5′-triphosphate(3TC).

Synthetic methods for the preparation of nucleosides and nucleotidesuseful in the present invention are likewise well known in the art asdisclosed in Acta Biochim. Pol., 43, 25-36 (1996); Swed. NucleosidesNucleotides 15, 361-378 (1996), Synthesis 12, 1465-1479 (1995),Carbohyd. Chem. 27, 242-276 (1995), Chem. Nucleosides Nucleotides 3,421-535 (1994), Ann. Reports in Med. Chem., Academic Press; and Exp.Opin. Invest. Drugs 4, 95-115 (1995).

The chemical reactions described in the references cited above aregenerally disclosed in terms of their broadest application to thepreparation of the compounds of this invention. Occasionally, thereactions may not be applicable as described to each compound includedwithin the scope of compounds disclosed herein. The compounds for whichthis occurs will be readily recognized by those skilled in the art. Inall such cases, either the reactions can be successfully performed byconventional modifications known to those skilled in the art, e.g., byappropriate protection of interfering groups, by changing to alternativeconventional reagents, by routine modification of reaction conditions,and the like, or other reactions disclosed herein or otherwiseconventional will be applicable to the preparation of the correspondingcompounds of this invention. In all preparative methods, all startingmaterials are known or readily preparable from known starting materials.

While nucleoside analogs are generally employed as antiviral agents asis, nucleotides (nucleoside phosphates) must sometimes have to beconverted to nucleosides in order to facilitate their transport acrosscell membranes. An example of a chemically modified nucleotide capableof entering cells is S-1-3-hydroxy-2-phosphonylmethoxypropyl cytosine(HPMPC, Gilead Sciences).

Immunomodulators and Immunostimulants

A large number of immunomodulators and immunostimulants that can be usedin the methods of the present invention are currently available. A listof these compounds is provided in Table 1, below.

TABLE 1 DRUG NAME AA-2G adamantylamide dipeptide adenosine deaminase,Enzon adjuvant, Alliance adjuvants, Ribi adjuvants, Vaxcel Adjuvaxagelasphin-11 AIDS therapy, Chiron algal glucan, SRI algammulin, AnutechAnginlyc anticellular factors, Yeda Anticort antigastrin-17 immunogen,Ap antigen delivery system, Vac antigen formulation, IDEC antiGnRHimmunogen, Aphton Antiherpin Arbidol Aviron azarole Bay-q-8939Bay-r-1005 BCH-1393 Betafectin Biostim BL-001 BL-009 BronocostatCantastim CDRI-84-246 cefodizime chemokine inhibitors, ICOS CMVpeptides, City of Hope CN-5888 cytokine-releasing agent, St DREAS,Paradigm DISC TA-KSV JO7B IO1A IO1Z ditiocarb sodium ECA-10-142 ELS-1endotoxin, Novartis FCE-20696 FCE-24089 FCE-24578 FLT-3 ligand, ImmunexFR-900483 FR-900494 FR-901235 FTS-Zn G-proteins, Cadus gludapcinglutaurine glycophosphopeptical GM-2 GM-53 GMDP growth factor vaccine,EntreM H-BIG, NABI H-CIG, NABI HAB-439 Helicobacter pylori vaccine,herpes-specific immune facto HIV therapy, United Biomed HyperGAM+CPImmuMax Immun BCG immune therapy, Connective immunomodulator, Evansimmunomodulators, Novacell imreg-1 imreg-2 Indomune inosine pranobexinterferon, Dong-A (alpha2) interferon, Genentech (gamma interferon,Novartis (alpha) interleukin-12, Genetics Ins interleukin-15, Immunexinterleukin-16, Research Cor ISCAR-1 J005x L-644257 licomarasminic acidLipoTher LK-409 LK-410 LP-2307 LT(R1926) LW-50020 MAF, Shionogi MDPderivatives, Merck met-enkephalin, TNI methylfurylbutyrolactones MIMPmirimostim mixed bacterial vaccine, Tem MM-1 moniliastat MPLA, RibiMS-705 murabutide murabutide, Vacsyn muramyl dipeptide derivativemuramyl peptide derivatives myelopid N-563 NACOS-6 NH-765 NISV, ProteusNPT-16416 NT-002 PA-485 PEFA-814 peptides, Scios peptidoglycan, PlivaPerthon, Advanced Plant PGM derivative, Pliva Pharmaprojects No. 1099Pharmaprojects No. 1426 Pharmaprojects No. 1549 Pharmaprojects No. 1585Pharmaprojects No. 1607 Pharmaprojects No. 1710 Pharmaprojects No. 1779Pharmaprojects No. 2002 Pharmaprojects No. 2060 Pharmaprojects No. 2795Pharmaprojects No. 3088 Pharmaprojects No. 3111 Pharmaprojects No. 3345Pharmaprojects No. 3467 Pharmaprojects No. 3668 Pharmaprojects No. 3998Pharmaprojects No. 3999 Pharmaprojects No. 4089 Pharmaprojects No. 4188Pharmaprojects No. 4451 Pharmaprojects No. 4500 Pharmaprojects No. 4689Pharmaprojects No. 4833 Pharmaprojects No. 494 Pharmaprojects No. 5217Pharmaprojects No. 530 pidotimod pimelautide pinafide PMD-589podophyllotoxin, Conpharm POL-509 poly-ICLC poly-ICLC, Yamasa ShoyuPolyA-PolyU Polysaccharide A protein A, Berlox Bioscience PS34W0pseudomonas MAbs, Teijin Psomaglobin PTL-78419 Pyrexol pyriferoneRetrogen Retropep RG-003 Rhinostat rifomaxil RM-06 Rollin romurtideRU-40555 RU-41821 rubella antibodies, RecCo S-27609 SB-73 SDZ-280-636SDZ-MRL-953 SX&F-107647 SLO4 SLO5 SM-4333 Solutein SRI-62-834 SRL-172ST-570 ST-789 staphage lysate Stimulon suppressin T-150R1 T-LCEFtabilautide temurtide Theradigm-HBV Theradigm-HPV Theradigm-HSV THF,Pharm & Upjohn THF, Yeda thymalfasin thymic hormone fractionsthymocartin thymolymphotropin thymopentin thymopentin analoguesthymopentin, Peptech thymosin fraction 5, Alpha thymostimilinthymotrinan TMD-232 TO-115 transfer factor, Viragen tuftsin, Sclavoubenimex Ulsastat ANGG− CD-4+ Collag+ COLSF+ COM+ DA-A+ GAST− GF-TH+GP-120− IF+ IF-A+ IF-A-2+ IF-B+ IF-G+ IF-G-1B+ IL-2+ IL-12+ IL-15+ IM+LHRH− LIPCOR+ LYM-B+ LYM-NK+ LYM-T+ OPI+ PEP+ PHG-MA+ RNA-SYN− SY-CW−TH-A-1+ TH-5+ TNF+ UNDosages

The N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds useful inthe present invention can be administered to humans in an amount in therange of from about 0.1 mg/kg/day to about 100 mg/kg/day, morepreferably from about 1 mg/kg/day to about 75 mg/kg/day, and mostpreferably from about 5 mg/kg/day to about 50 mg/kg/day.

The nucleoside or nucleotide antiviral compound, or mixtures thereof,can be administered to humans in an amount in the range of from about0.1 mg/person/day to about 500 mg/person/day, preferably from about 10mg/person/day to about 300 mg/person/day, more preferably from about 25mg/person/day to about 200 mg/person/day, even more preferably fromabout 50 mg/person/day to about 150 mg/person/day, and most preferablyin the range of from about 1 mg/person/day to about 50 mg/person/day.

Immunomodulators and immunostimulants useful in the present inventioncan be administered in amounts lower than those conventional in the art.For example, thymosin alpha 1 and thymosin fraction 5 are typicallyadministered to humans for the treatment of HepB infections in an amountof about 900 μg/m², two times per week (Hepatology (1988) 8:1270;Hepatology (1989) 10:575; Hepatology (1991) 14:409; Gastroenterology(1995) 108:A1127). In the methods and compositions of the presentinvention, this dose can be in the range of from about 10 μg/m² twotimes per week to about 750 μg/m², two times per week, more preferablyfrom about 100 μg/m², two times per week to about 600 μg/m², two timesper week, most preferably from about 200 μg/m², two times per week toabout 400 μg/m², two times per week. Interferon alfa is typicallyadministered to humans for the treatment of HepC infections in an amountof from about 1×10⁶ units/person, three times per week to about 10×10⁶units/person, three times per week (Simon et al., (1997) Hepatology25:445-448). In the methods and compositions of the present invention,this dose can be in the range of from about 0.1×10⁶ units/person, threetimes per week to about 7.5×10⁶ units/person, three times per week, morepreferably from about 0.5×10⁶ units/person, three times per week toabout 5×10⁶ units/person, three times per week, most preferably fromabout 1×10⁶ units/person, three times per week to about 3×10⁶units/person, three times per week.

Due to the enhanced hepatitis virus antiviral effectiveness of theseimmunomodulators and immunostimulants in the presence of theN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds useful in thepresent invention, reduced amounts of otherimmunomodulators/immunostimulants can be employed in the methods andcompositions disclosed herein. Such reduced amounts can be determined byroutine monitoring of hepatitis virus in infected patients undergoingtherapy. This can be carried out by, for example, monitoring hepatitisviral DNA in patients' serum by slot-blot, dot-blot, or PCR techniques,or by measurement of hepatitis surface or other antigens, such as the eantigen, in serum. Methods therefor are discussed in Hoofnagle et al.,(1997) New Engl. Jour. Med. 336(5):347-356, and F. B. Hollinger inFields Virology, Third Ed., Vol. 2 (1996), Bernard N. Fields et al.,Eds., Chapter 86, “Hepatitis B Virus,” pp. 2738-2807, Lippincott-Raven,Philadelphia, Pa., and the references cited therein.

Patients can be similarly monitored during combination therapy employingN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds and nucleosideand/or nucleotide antiviral agents to determine the lowest effectivedoses of each.

The doses described above can be administered to a patient in a singledose or in proportionate multiple subdoses. In the latter case, dosageunit compositions can contain such amounts of submultiples thereof tomake up the daily dose. Multiple doses per day can also increase thetotal daily dose should this be desired by the person prescribing thedrug.

Pharmaceutical Compositions

The compounds of the present invention can be formulated aspharmaceutical compositions. Such compositions can be administeredorally, parenterally, by inhalation spray, rectally, intradermally,transdermally, or topically in dosage unit formulations containingconventional nontoxic pharmaceutically acceptable carriers, adjuvants,and vehicles as desired. Topical administration may also involve the useof transdermal administration such as transdermal patches oriontophoresis devices. The term parenteral as used herein includessubcutaneous, intravenous, intramuscular, or intrasternal injection, orinfusion techniques. Formulation of drugs is discussed in, for example,Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa. (1975), and Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. (1980).

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid are usefulin the preparation of injectables. Dimethyl acetamide, surfactantsincluding ionic and non-ionic detergents, and polyethylene glycols canbe used. Mixtures of solvents and wetting agents such as those discussedabove are also useful.

Suppositories for rectal administration of the compounds discussedherein can be prepared by mixing the active agent with a suitablenon-irritating excipient such as cocoa butter, synthetic mono-, di-, ortriglycerides, fatty acids, or polyethylene glycols which are solid atordinary temperatures but liquid at the rectal temperature, and whichwill therefore melt in the rectum and release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, thecompounds of this invention are ordinarily combined with one or moreadjuvants appropriate to the indicated route of administration. Ifadministered per os, the compounds can be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, cellulose alkylesters, talc, stearic acid, magnesium stearate, magnesium oxide, sodiumand calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled-release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering agents such as sodium citrate, or magnesiumor calcium carbonate or bicarbonate. Tablets and pills can additionallybe prepared with enteric coatings.

For therapeutic purposes, formulations for parenteral administration canbe in the form of aqueous or non-aqueous isotonic sterile injectionsolutions or suspensions. These solutions and suspensions can beprepared from sterile powders or granules having one or more of thecarriers or diluents mentioned for use in the formulations for oraladministration. The compounds can be dissolved in water, polyethyleneglycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil,sesame oil, benzyl alcohol, sodium chloride, and/or various buffers.Other adjuvants and modes of administration are well and widely known inthe pharmaceutical art.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents is commonly used in the art, such as water.Such compositions can also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

The amount of active ingredient that can be combined with the carriermaterials to produce a single dosage form will vary depending upon thepatient and the particular mode of administration.

Certain of the pharmaceutical compounds of this invention which areadministered in accordance with the methods of the invention can serveas prodrugs to other compounds of this invention. Prodrugs are drugsthat can be chemically converted in vivo or in vitro by biologicalsystems into an active derivative or derivatives. Prodrugs areadministered in essentially the same fashion as the other pharmaceuticalcompounds of the invention. Non-limiting examples are the esters of theN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds of thisinvention.

Treatment Regimen

The regimen for treating a patient suffering from a hepatitis virusinfection with the compounds and/or compositions of the presentinvention is selected in accordance with a variety of factors, includingthe age, weight, sex, diet, and medical condition of the patient, theseverity of the infection, the route of administration, pharmacologicalconsiderations such as the activity, efficacy, pharmacokinetic, andtoxicology profiles of the particular compounds employed, and whether adrug delivery system is utilized.

Administration of the drug combinations disclosed herein shouldgenerally be continued over a period of several weeks to several monthsor years until virus titers reach acceptable levels, indicating thatinfection has been controlled or eradicated. As noted above, patientsundergoing treatment with the drug combinations disclosed herein can beroutinely monitored by measuring hepatitis viral DNA in patients' serumby slot-blot, dot-blot, or PCR techniques, or by measurement ofhepatitis antigens, such as hepatitis B surface antigen (HBsAg) andhepatitis B e antigen (HBeAg), in serum to determine the effectivenessof therapy. In chronic hepatitis B, for example, remissions arecharacterized by the disappearance of hepatitis B viral DNA, i.e.,reduction to undetectable levels as measured by hybridization testscapable of detecting levels ≧10⁵ genomes per ml of serum, and HBeAg fromserum despite the continued presence of HBsAg. These serologic eventsare followed by improvement in the biochemical and histologic featuresof the disease. The end point of successful treatment in most trials ofantiviral therapy is the disappearance of HBeAg and viral DNA fromserum. In patients in whom the e antigen disapppears, remission isusually sustained, and results in an inactive HBsAg carrier state. Manypatients eventually become HBsAg-negative (see Hoofnagle et al., (1997)New Engl. Jour. Med. 336(5):347-356 for a review).

Continuous analysis of the data obtained by these methods permitsmodification of the treatment regimen during therapy so that optimalamounts of each component in the combination are administered, and sothat the duration of treatment can be determined as well. Thus, thetreatment regimen/dosing schedule can be rationally modified over thecourse of therapy so that the lowest amounts of each of the antiviralcompounds used in combination which together exhibit satisfactoryanti-hepatitis virus effectiveness are administered, and so thatadministration of such antiviral compounds in combination is continuedonly so long as is necessary to successfully treat the infection.

The following non-limiting examples serve to illustrate various aspectsof the present invention.

EXAMPLE 1 Preparation of 1,5-(butylimino)-1,5-dideoxy-D-glucitol

A solution of 1,5-dideoxy-1,5-imino-D-glucitol (5.14 g, 0.0315 mole),butyraldehyde (3.35 ml, 0.0380 mole) and Pd black (1 g) in 200 mlmethanol was hydrogenated (60 psi/29° C./21 hrs.). After filtering theresulting mixture, the filtrate was concentrated in vacuo to an oil. Thetitle compound was crystallized from acetone, and recrystallized frommethanol/acetone, m.p. ca. 132° C. The structure assignment wassupported by NMR, infrared spectra and elemental analysis.

Analysis calcd. for C₁₀H₂₁NO₄: C, 54.78; H, 9.65; N, 6.39. Found: C,54.46; H, 9.33; N, 6.46.

EXAMPLE 2 Preparation of 1,5-(butylimino)-1,5-dideoxy-D-glucitol,tetraacetate

Acetic anhydride (1.08 g, 0.0106 mole) was added to the title compoundof Example 1 (0.50 g, 0.0023 mole) in 5 ml pyridine and stirred for 17days at room temperature. The product was evaporated under nitrogen gas.The resulting title compound was purified by silica gel chromatography.Structure assignment was supported by NMR, infrared spectra andelemental analysis.

Analysis calcd. for C₁₈H₂₉NO₈: C, 55.80; H, 7.54; N, 3.62. Found: C,55.42; H, 7.50; N, 3.72.

EXAMPLE 3 Anti-Hepatitis B Virus Activity of(−)-2′-deoxy-3′-thiocytidine-5′-triphosphate (3TC) Alone and inCombination with N-nonyl-DNJ

The anti-hepatitis B virus effect of(−)-2′-deoxy-3′-thiocytidine-5′-triphosphate (3TC) alone and incombination with N-nonyl-DNJ was determined according to Korba ((1996)Antiviral Research 29(1):49-51), using the “combostat” strategy (ComstatProgram, Combostat Corp., Duluth, Minn.). The combostat method involvesserially diluting the IC-90 of each compound. The IC-90 of N-nonyl-DNJhas been determined to be between 4 and 10 μg/ml (T. Block and G. Jacob,unpublished observation). The accepted IC-90 for 3TC in HepG 2.2.15(2.2.15) cells is 300 nM to 500 nM (Doong et al. (1991) Proc. Natl.Acad. Sci. USA 88:8495-8499).

2.2.15 cells, described in Sells et al. (1987) Proc. Natl. Acad. Sci.USA 84:1005-1009, were maintained in RPMI 1640 medium (Gibco BRL,#31800-022) supplemented with 10% fetal bovine serum, 200 μg/ml G418(Gibco BRL 066-1811). Cells were seeded into 25 cm² flasks at 80%confluency. Five days later, flasks in triplicate received either nocompound, serial dilutions of 3TC alone, or serial dilutions of 3TC plusN-nonyl-DNJ. At 2, 4, and 6 days after addition of compound (with mediumreplacement on those days), the amount of hepatitis B virus (HBV) DNA inthe culture medium was determined by PCR analysis ofpolyethyleneglycol-sedimented particles. Thus, in these experiments,enveloped particles were not distinguished from nucleocapsids.PCR-amplified products were resolved by agarose gel electrophoresis(1.5% agarose), and the 538 nucleotide fragment was quantified by bandscanning (HP Jet Imager). The amount of HBV recovered from untreatedcells is assumed to be 100%. Data from the 6-day time point arepresented in FIG. 1 as the average values from at least three separateflasks, and the standard error was never greater than 20%, with anaverage error of 12%.

For each of the three time point series tested, the combination of 3TCplus N-nonyl-DNJ was significantly more effective in inhibiting HBVsecretion than either compound alone. Conclusions based upon PCRanalysis alone make it difficult to assign precise IC-50 values. Theextreme sensitivity and delicate nature of PCR, for example, may accountfor the inability to achieve greater than 90% inhibition of HBV by 3TCalone, even at 300 nM. Every experiment included controls to assure thatPCR was performed in a range of concentrations of DNA in which thereaction yields results proportional to the amount of DNA in the sample.Resolution is approximately 3-fold, i.e., 3-fold differences in DNAconcentrations can be detected. The inability to consistently detectless than 3-fold differences probably explains the failure of 3TC aloneto achieve 90% inhibition. This suggests that a very high standard ofinhibition must be met for the PCR to detect inhibition. Consequently,the trend, over three separate time points, is clear: the combinedeffect of 3TC plus N-nonyl-DNJ is greater than that of either compoundalone, or the additive individual effects of each compound. These datasuggest that the IC-50 of 3TC has been moved from about 60 nM to about0.48 nM when 0.016 μg/ml N-nonyl-DNJ is present.

EXAMPLE 4 Anti-Hepatitis B Virus Effect of N-nonyl-DNJ Alone in aWoodchuck Model

In order to evaluate the efficacy of N-nonyl-DNJ in combination with 3TC(or other nucleoside or nucleotide analogs) against Hepatitis B virus ina woodchuck animal model, an monotherapy experiment using N-nonyl-DNJalone was first conducted. This was necessary to determine ifN-nonyl-DNJ has any anti-HBV effect in the woodchuck and, if N-nonyl-DNJhas a beneficial effect, to design a combination study based on thedose-response relationship of this drug alone.

Therefore, five groups of four animals each (all groups had both sexes,all but the control had two of each sex) were assigned to 0, 12.5, 25,50, and 100 mg/kg/day with BID oral dosing. These were lab-reared wildanimals. All animals were infected with woodchuck hepatitis virus (WHV)as neonates, and had been tested positive on serological tests for WHVsurface antigen. Blood samples were drawn one week prior to dosing (−1week), immediately before dosing (0 weeks), weekly during dosing (1, 2,3, and 4 weeks), and after the end of dosing (5, 6, 8, and 10 weeks).

There are two measures of drug efficacy: reduction in total HBV DNA(measured by quantitative PCR), and reduction in HBV DNA from capsidswith intact surface glycoproteins, which is the active form of the virus(measured by an ELISA-like immune precipitation assay followed byquantitative PCR). Cell culture experiments with N-nonyl-DNJdemonstrated little or no effect of this compound on total HBV DNA, buta marked effect on the immune precipitated DNA (IPDNA). Notsurprisingly, the IPDNA assay is quite variable; as a partialcompensation for this, four assay runs were conducted, each containingsamples from all animals, but different subsets of the study weeks.

To summarize the results, N-nonyl-DNJ had no effect on total HBV DNAmeasurements, which were essentially constant for all dose levels overthe pre-dose and dosed portions of the study. On the other hand, IPDNAlevels were not constant over the study period. The low dose animalstended to have increasing levels of IPDNA over the dosing period (weeks0-4), while high dose animals tended to have decreasing levels of IPDNAover the same period. Fitting a straight line to each animal's weeklyresponses gave a significant difference in the slope of these lines dueto either dose or plasma level of drug. The plasma levels of drug werealso quite variable: animals with the lowest plasma levels in their dosegroup had lower plasma levels than the animals with the highest plasmalevels from the next lower dose group. There were no differences betweenresponses of males and females on any of the measures.

Plasma Levels

There were no clear patterns in the changes in plasma levels ofN-nonyl-DNJ which could be related to week of dosing or time sinceprevious dose. Because the plasma levels within an animal seemedreasonably consistent during dosing, the median plasma level for eachanimal was used for subsequent modeling. The plasma levels for each weekof the dosing-period are plotted for each animal vs. dose (a smallamount of random noise is added to the dose level so points which wouldlie on top of each other on the plot can be distinguished) (FIG. 2).

HBV DNA

The total HBV DNA levels were essentially constant over time within eachanimal (data not shown). There was a faint hint of a dose-responserelationship with decreasing levels of virus with increasing levels ofdrug, except that three animals at the highest dose had very high viruslevels. It is not possible to conclude that there is any relationshipbetween dose of N-nonyl-DNJ and total HBV DNA. It is possible that thereare two populations of animals, responders (such as animal r) andnon-responders (animals i, m, and d), but more data would be required topermit a firm conclusion on this point.

Immune Precipitated HBV DNA

Substantial variation existed in the IPDNA assay, both between assayruns and within assay runs (data not shown). Even so, it was possible toobserve and model a slope over weeks 0-4 which is generally increasingfor low dose animals and decreasing for high dose animals. This changein slope was statistically significant (p<0.005).

Before models are fitted to the data, a log transform was appliedbecause: 1) the variation in IPDNA increases with increasing IPDNAvalues; the log transformation gives values with a nearly constantvariation, and 2) it is expected that drug effects will appear as aconstant multiplier of the IPDNA level. Because there are zero values ofIPDNA, a small value (about ½ of the smallest non-zero value) was addedto all values before the log transform.

Two approaches were used to model the changes in slope to week with doseof N-nonyl-DNJ: a linear modeling approach and a nonlinear model. Bothapproaches assume that the (linear) rate of change of the Log(IPDNA)measure over the dosing period is the “right” measure to reflect theeffect of the drug on the virus. Both approaches are fit in stages, andthe first stage is common to both approaches. First, a simple straightline regression model is fit using weeks 0-4 to predict log(IPDNA+10)separately for each animal by run combination. In the second stage, theresponse variable is the slope fitted in the first stage.

For the linear approach, a model is fit with slope to week as theresponse where run is considered a block, dose has a significant effect(almost all of this effect is due to a slope to dose), and the relevanterror for testing the effect of dose is the variation among animalstreated alike (after the adjustment for the runs as blocks). This issimilar to using the calibration data within each run to first adjusteach run's data to a common virus DNA concentration; the difference isthat here the data from the woodchucks are used for the run adjustmentrather than only the calibration data.

For the nonlinear approach, a four parameter logistic model is fit withthe slope to week as the response and the dose as the predictor. Again,run is considered a block, but because no run has all weeks, it is notpossible to fully reflect the blocking in the nonlinear approach. Evenso, the nonlinear model yields an EC50 of 7.88 mg/kg/BID dose. Theaverage maximum slope observed was 2.71 additional Log(IPDNAμg/mL)/week, or an increase of about 150%/week, the average minimumslope observed with N-nonyl-DNJ is 0.31 fewer Log(IPDNA μg/mL)/week), orabout a decrease of about 25%/week. The slopes, the fitted model, theparameter estimates from the model, and the approximate standard errorsfor these parameters are all shown in FIG. 3. The data indicate anapproximate effective monotherapy dose of N-nonyl-DNJ in woodchucks ofabout 16 mg/kg/day. Whether in woodchucks or humans, the effective doseof both the N-alkyl-DNJ and nucleoside or nucleotide antiviral agentadministered in combination therewith can be administered in two equaldaily subdoses (i.e., B.I.D.).

FIGS. 2 and 3 show letters to indicate animals. Table 2 shows two of theanimal codes, the sex, and the dose.

TABLE 2 Animal Codes, Sex, and Dose Animal Number Letter Code Sex DoseF95343 b F 0 M96364 n M 0 F96304 k F 0 F96301 j F 0 M96285 h M 6.25F96283 g F 6.25 F96391 o F 6.25 M96305 l M 6.25 F96271 f F 12.5 M96256 eM 12.5 M96404 s M 12.5 F96392 p F 12.5 F96163 c F 25 M96414 t M 25F96393 q F 25 M95322 a M 25 M96286 i M 50 F96231 d F 50 F96402 r F 50M96363 m M 50

EXAMPLE 5 Antiviral Study to Test the Activity of N-nonyl-DNJ inCombination with 3TC in a Woodchuck Model of Hepatitis B Virus Infection

The combined activity of N-nonyl-DNJ and the nucleoside analog 3TC canbe assessed using the woodchuck model of hepatitis B virus infection.Twenty-eight woodchucks with persistent woodchuck hepatitis virus (WHV)infection can be utilized. Groups of woodchucks can be treated orallywith 3TC alone (s.i.d.), with N-nonyl-DNJ alone (b.i.d.), or withcombinations of the two drugs. The antiviral activity of the individualdrugs and combinations can be assessed by measuring serum WHV DNA duringtreatment, and comparing the results of treated groups to placebotreated controls.

Twenty-eight woodchucks with established persistent WHV infection can beused, all of which were experimentally infected with WHV during thefirst week of life. All can be WHsAg positive at the time the study isinitiated.

A total of eight experimental groups can be used. Woodchucks in eachgroup can be stratified on the basis of gender, body weight, and age.3TC can be administered orally as an aqueous-suspension of Epivir(Glaxo-Wellcome) tablets one time per day. N-nonyl-DNJ can also beadministered orally in aqueous solution, in two divided doses. Treatmentwith both drugs can be followed by the administration of 4 to 5 mls ofsemisynthetic liquid woodchuck diet to insure complete ingestion of thedrugs.

The experimental groups can be as follows:

Group 3TC N-nonyl-DNJ ID No. (mg/kg/day) (mg/kg/day) 1 4 0.0 0.0 2 3 3.00.0 3 3 9.0 0.0 4 3 0.0 4.0 5 3 0.0 12.0 6 4 1.5 2.0 7 4 4.5 6.0 8 4 9.012.0

Woodchucks can be anesthetized (50 mg/kg ketamine, 5 mg/kg zylazine),weighed, and blood samples obtained prior to initial treatment, atweekly intervals during the six week period of treatment, and at 1, 2,and 4 weeks following treatment. Serum can be harvested and divided intoaliquots. One aliquot can be used for analysis of WHV DNA by dot blothybridization and for WHsAg by ELISA. CBCs and clinical biochemicalprofiles can be obtained prior to treatment and at the end of treatment.A second aliquot can be maintained as an archive sample. Other aliquotsof serum can be used for drug analysis and special WHV DNA analyses.

The invention being thus described, it will be obvious that the same canbe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the present invention, and allsuch modifications and equivalents as would be obvious to one skilled inthe art are intended to be included within the scope of the followingclaims.

1. A pharmaceutical composition for treating a hepatitis B virusinfection in a human patient, comprising from about 0.1 mg to about 100mg of N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol and from about 0.1mg to about 500 mg of (−)-2′-deoxy-3′-thiocytidine-5′-triphosphate.
 2. Apharmaceutical composition for treating hepatitis virus infection,comprising a first amount of anN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula I:

wherein R is selected from the group consisting of straight chain alkylhaving a chain length of C₇ to C₁₂ and W, X, Y, and Z are eachindependently selected from the group consisting of hydrogen, andbutanoyl; and a second amount of an antiviral compound selected from thegroup consisting of 2′,3′-dideoxycytidine,2′,3′-dideoxycytidine-5′-triphosphate,(−)-2′,3′-dideoxy-3′-thiacytidine,(−)-2′-deoxy-3′-thiocytidine-5′-triphosphate, and mixtures thereof and apharmaceutical acceptable carrier, diluent, or excipient wherein saidfirst and second amounts of said compounds together comprise ananti-hepatitis virus effective amount of said compounds.
 3. Thepharmaceutical composition of claim 2, wherein R is a straight chainalkyl having a chain length of C₇ to C₁₂ and W, X, Y, and Z are eachhydrogen.
 4. The pharmaceutical composition of claim 3, wherein R isnonyl.
 5. The pharmaceutical composition of claim 2, wherein R is nonyland W, X, Y, and Z are each butanoyl.
 6. The pharmaceutical compositionof claim 2, wherein said N-substituted-1,5-dideoxy-1,5-imino-D-glucitolcompound is selected from the group consisting ofN-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol andN-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate, and saidantiviral compound is (−)-2′-deoxy-3′-thiocytidine-5′-triphosphate. 7.The pharmaceutical composition of claim 6, wherein saidN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound isN-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol.
 8. The pharmaceuticalcomposition of claim 2, wherein said first amount of saidN-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound is in the rangeof from about 0.1 mg to about 100 mg.
 9. The pharmaceutical compositionof claim 2, wherein said second amount of said nucleoside or nucleotideantiviral compound, or mixture thereof, is in the range of from about0.1 mg to about 500 mg.